Use of 3-(5-amino-2-methyl-4-oxoquinazolin-3(4h)-yl)piperidine-2,6-dione in treatment of immune-related and inflammatory diseases

ABSTRACT

Provided herein are methods of using compounds and compositions for modulating lymphocytic activity, including activity of B cells and/or T cells, in immune-related diseases or inflammatory diseases. Pharmaceutical compositions and dosing regimens for use in the methods are also provided herein.

1. CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Patent ApplicationNo. 61/451,995, filed on Mar. 11, 2011, and U.S. Provisional PatentApplication No. 61/480,272, filed on Apr. 28, 2011, which are herebyincorporated by reference herein in their entireties.

2. SEQUENCE LISTING

The present application is being filed with a Sequence Listing submittedas filename 12827-263-228_SeqListing.txt, of size 6,571 bytes, which wascreated on Mar. 8, 2012. The Sequence Listing is incorporated herein byreference in its entirety.

3. FIELD

Provided herein are methods of treating, preventing, and/or managingdiseases associated with lymphocytic activity, including activity of Bcells and/or T cells, e.g., immune-related diseases or inflammatorydiseases, comprising administering Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer, racemicmixture, co-crystal, clathrate, or polymorph thereof, where Compound Iis 3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.Pharmaceutical compositions and dosing regimens for such treatment,prevention, and/or management are also provided herein.

4. BACKGROUND

Inflammatory and immune-related diseases modulated by lymphocyticactivity, including activity of B cells and/or T cells, such as lupus,scleroderma, Sjögren syndrome, ANCA-induced vasculitis,anti-phospholipid syndrome and myasthenia gravis, continue to beimportant medical problems.

Lupus or lupus erythematosus is a collection of autoimmune disordersthat can cause chronic inflammation in various parts of the body,especially the skin, joints, blood, and kidneys. The body's immunesystem normally makes proteins called antibodies to protect the bodyagainst viruses, bacteria, and other foreign materials (i.e., antigens).In an autoimmune disorder such as lupus, the immune system loses itsability to tell the difference between antigens and its own cells andtissues and can make antibodies directed against its own cells andtissues to form immune complexes. These immune complexes can build up inthe tissues and cause inflammation, injury to tissues and/or pain. Thethree most common types of lupus include systemic lupus erythematosus(SLE), cutaneous lupus erythematosus (CLE) and drug-induced lupus. Moredetailed descriptions of lupus or lupus erythematosus can be found inWallace, 2000, The Lupus Book: A Guide for Patients and Their Families,Oxford University Press, Revised and Expanded Edition, which isincorporated by reference herein in its entirety.

Scleroderma is a rare disease with a stable incidence of approximately19 cases per 1 million persons. The exact cause of scleroderma isunknown. Abnormalities involve autoimmunity and alteration ofendothelial cell and fibroblast function. Systemic scleroderma usuallybegins with skin thickening, usually of the fingers, accompanied byRaynaud's phenomenon. Raynaud's disease typically precedes furthermanifestations of systemic scleroderma. Early in the disease theaffected skin may be puffy and soft. The usual location of greatest skinthickening and hardening is the face, hands and fingers. Sclerodactylyis frequently present. Tendon friction rubs are often palpable on examand can be painful. With more advanced disease, digital ulcers andauto-amputation may occur. Gastrointestinal dismotility is a feature,often manifested by heartburn, or by diarrhea with malabsorption orpseudo-obstruction. New onset hypertension or renal insufficiency aremanifestations of the associated vascular injury. Heart failure orarrhythmia are also possible due to cardiac fibrosis. (Hachulla E,Launay D, Diagnosis and classification of systemic sclerosis, Clin RevAllergy Immunol 2010; 40(2):78-83).

The major manifestations of scleroderma and in particular of systemicsclerosis are inappropriate excessive collagen synthesis and deposition,endothelial dysfunction, spasm, collapse and obliteration by fibrosis.In terms of diagnosis, an important clinical parameter is skinthickening proximal to the metacarpophalangeal joints. Raynaud'sphenomenon is a frequent, almost universal component of scleroderma. Itis diagnosed by color changes of the skin upon cold exposure. Ischemiaand skin thickening are symptoms of Raynaud's disease.

There remains a need for prophylactic or therapeutic drugs that can beused to treat or prevent immune-related and inflammatory diseases,including lupus, scleroderma, Sjögren syndrome, ANCA-induced vasculitis,anti-phospholipid syndrome and myasthenia gravis.

5. SUMMARY

Provided herein are methods of treating, managing, ameliorating and/orpreventing diseases, disorders and/or conditions associated withimmune-related and inflammatory diseases comprising administering atherapeutically effective amount of a compound of formula I

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer, racemic mixture, co-crystal, clathrate, or polymorph thereof

In one embodiment, the compound is3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

In certain embodiments, the disease is selected from lupus, scleroderma,Sjögren syndrome, ANCA-induced vasculitis, anti-phospholipid syndromeand myasthenia gravis.

In one embodiment, provided herein are methods of modulating, e.g.,reducing, lymphocytic activity, including activity of B cells and/or Tcells, comprising contacting B cell and/or T cell with an effectiveamount of Compound I.

Also provided herein are pharmaceutical compositions, single unit dosageforms, and kits suitable for use in treating, preventing, amelioratingand/or managing diseases, disorders and/or conditions associatedimmune-related and inflammatory diseases, which comprise Compound I,optionally in combination with one or more other therapeutic agents.

In certain embodiments, Compound I is administered in combination withone or more therapeutic agents, i.e., pharmaceutical agents that aremodulators of lymphocytic activity, including activity of B cells and/orT cells activity. The combinations encompass simultaneous as well assequential administration.

6. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the effect of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione oncytokine and chemokine production in anti-CD3-stimulated human T cells,expressed as absolute amount produced.

FIG. 2 illustrates the effect of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione oncytokine and chemokine production in anti-CD3-stimulated human T cells,expressed as percentage of control.

FIG. 3 illustrates inhibition of production of cytokine and chemokineproduction in lipopolysaccharide-stimulated peripheral blood mononuclearcells by3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

FIG. 4 illustrates enhancement of production of cytokine and chemokineproduction in lipopolysaccharide-stimulated peripheral blood mononuclearcells by3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

FIG. 5 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as absolute amountproduced, for3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

FIG. 6 illustrates enhancement of NK cell IFN-gamma production inresponse to immobilized IgG and IL-2, expressed as percentage of amountof IFN-gamma produced in the presence of 1 μm pomalidomide, for3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

FIG. 7 illustrates the effect of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione onNK-cell mediated ADCC against Rituximab coated lymphoma cells.

FIG. 8 illustrates hematoxylin and eosin stained skin sectionphotomicrographs showing dermal thickness of lesional skin in thebleomycin dermal fibrosis mouse model (prevention of inflammation drivenfibrosis).

FIG. 9 illustrates hematoxylin and eosin stained skin sectionphotomicrographs showing dermal thickness of lesional skin in thebleomycin dermal fibrosis mouse model (regression of establishedfibrosis).

7. DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications are incorporated by reference in their entirety. In theevent that there is a plurality of definitions for a term herein, thosein this section prevail unless stated otherwise.

As used herein, and unless otherwise indicated, the terms “treat,”“treating” and “treatment” refer to alleviating or reducing the severityof a disease or a symptom associated with the disease or condition beingtreated.

As used herein, “prevent”, “prevention” and other forms of the wordinclude the inhibition of onset or progression of a disease or disorderor a symptom of the particular disease or disorder. In some embodiments,subjects with familial history of cancer are candidates for preventiveregimens. Generally, in the context of cancer, the term “preventing”refers to administration of the drug prior to the onset of signs orsymptoms of a cancer, particularly in subjects at risk of cancer.

As used herein, and unless otherwise indicated, the term “managing”encompasses preventing the recurrence of the particular disease ordisorder in a subject who had suffered from it, lengthening the time asubject who had suffered from the disease or disorder remains inremission, reducing mortality rates of the subjects, and/or maintaininga reduction in severity or avoidance of a symptom associated with thedisease or condition being managed.

As used herein, “subject” means an animal, typically a mammal, includinga human being. As used herein, “patient” means a human subject.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundrefer to an amount sufficient to provide a therapeutic benefit in thetreatment, prevention and/or management of a disease, to delay orminimize one or more symptoms associated with the disease or disorder tobe treated. The terms “therapeutically effective amount” and “effectiveamount” can encompass an amount that improves overall therapy, reducesor avoids symptoms or causes of disease or disorder or enhances thetherapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, the term“prophylactically effective amount” of a compound is an amountsufficient to prevent a disease or condition, or one or more symptomsassociated with the disease or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease. Theterm “prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” includes, but is not limited to, asalt of an acidic group that can be present in the compounds providedherein. Under certain acidic conditions, the compound can form a widevariety of salts with various inorganic and organic acids. The acidsthat can be used to prepare pharmaceutically acceptable salts of suchbasic compounds are those that form salts comprising pharmacologicallyacceptable anions including, but not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, bromide, iodide, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydroxynaphthoate, isethionate, lactate, lactobionate,malate, maleate, mandelate, methanesulfonate (mesylate), methylsulfate,muscate, napsylate, nitrate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, succinate, sulfate, tannate,tartrate, teoclate, triethiodide, and pamoate.

As used herein and unless otherwise indicated, the term “hydrate” meansa compound provided herein or a salt thereof, further including astoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces. The hydrates can be crystalline ornon-crystalline.

As used herein and unless otherwise indicated, the term “solvate” meansa solvate formed from the association of one or more solvent moleculesto compound provided herein. The term “solvate” includes hydrates (e.g.,monohydrate, dihydrate, trihydrate, tetrahydrate, and the like). Thesolvates can be crystalline or non-crystalline.

As used herein, and unless otherwise specified, the term “stereoisomer”encompasses all enantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds provided herein.

As used herein, and unless otherwise indicated, the term“stereomerically pure” or “enantiomerically pure” means that a compoundcomprises one stereoisomer and is substantially free of its counterstereoisomer or enantiomer. For example, a compound is stereomericallyor enantiomerically pure when the compound contains 80%, 90%, or 95% ormore of one stereoisomer and 20%, 10%, or 5% or less of the counterstereoisomer. In certain cases, a compound provided herein is consideredoptically active or stereomerically/enantiomerically pure (i.e.,substantially the R-form or substantially the S-form) with respect to achiral center when the compound is about 80% ee (enantiomeric excess) orgreater, preferably, equal to or greater than 90% ee with respect to aparticular chiral center, and more preferably 95% ee with respect to aparticular chiral center.

As used herein, and unless otherwise indicated, the term“stereomerically enriched” or “enantiomerically enriched” encompassesracemic mixtures as well as other mixtures of stereoisomers of compoundsprovided herein (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40,65/35 and 70/30).

The terms “co-administration” and “in combination with” include theadministration of two or more therapeutic agents (for example, CompoundI or a composition provided herein and another modulator of lymphocyticactivity, including activity of B cells and/or T cells activity or otheractive agent) either simultaneously, concurrently or sequentially withno specific time limits. In one embodiment, Compound I and at least oneother agent are present in the cell or in the subject's body at the sametime or exert their biological or therapeutic effect at the same time.In one embodiment, the therapeutic agent(s) are in the same compositionor unit dosage form. In another embodiment, the therapeutic agent(s) arein separate compositions or unit dosage forms.

A “B cell” is a lymphocyte that matures within the bone marrow, andincludes a naive B cell, memory B cell, or effector B cell (plasmacells). The B cell herein may be a normal or non-malignant B cell.

A “T cell” is a lymphocyte that matures in thymus, and includes a helperT cell, a memory T cell, and a cytotoxic T cell.

As used herein “overall survival” refers to the time from randomizationuntil death from any cause, and is measured in the intent-to-treatpopulation. Overall survival can be evaluated in randomized controlledstudies.

As used herein “objective response rate” refers to the proportion ofpatients with reduced predefined scleroderma symptoms at the end of apredefined period of time. Response duration is usually measured fromthe time of initial response until documented scleroderma progression.

As used herein “time to progression” means the time from randomizationuntil objective scleroderma progression. In certain embodiments, time toprogression does not include deaths.

As used herein “progression-free survival” means the time fromrandomization until objective scleroderma progression or death.

As used herein “time-to-treatment failure” means any endpoint(s)measuring time from randomization to discontinuation of treatment forany reason, including disease progression, treatment toxicity, anddeath.

As used herein “mortality” means a measure of the number of deaths in agiven population.

As used herein “respiratory mortality” means patients who die from acutehypoxemia or other specific respiratory deterioration resulting in deathsuch as need for mechanical ventilation leading to death, respiratoryarrest, or any other event in a subject deemed to be respiratory innature.

As used herein “respiratory hospitalization” means those hospitalizedfor deterioration in pulmonary status as documented by patient hospitaladmission notes or other medical opinion.

As used herein “modified Rodnan skin score” means a validated numericalscoring system to assess dermal skin thickness.

As used herein “skin thickness” means hard or indurated skin that can beevaluated using a variety of techniques including durometer and mRSS

As used herein “skin induration” means skin that is hardened, red,inflamed, thickened or tender.

As used herein “dermatology quality of life index” means an evaluationof quality or life related to the skin symptoms for a patient havingscleroderma.

As used herein “pulmonary function” means any measurement of forcedexpiratory flow, forced vital capacity, FEV 25-75%, lung volumes orvital capacity.

As used herein “carbon monoxide diffusing capacity” means an assessmentof the uptake of carbon monoxide across the alveolar-capillary membrane.It can be a proxy for the measurement of the lungs ability to transferoxygen from the lungs to the blood stream.

As used herein “Mahler Dyspnea index” means an instrument that providesclinical measurement of shortness of breath.

As used herein “Saint George's Respiratory Questionnaire score” means aninstrument that measures quality of life in patients with pulmonarydisease.

As used herein “UCLA scleroderma clinical trial consortiumgastrointestinal tract score” means a questionnaire administered topatients having scleroderma to evaluate gastrointestinal symptomsassociated with scleroderma (systemic sclerosis).

As used herein “flow-mediated dilatation” means any measurement ofvascular endothelial function in a patient having scleroderma.

As used herein “six minute walk distance” means any evaluation of thedistance a patient having scleroderma can walk within 6 minutes or anystandardized procedure to evaluate ability to walk for a fixed period oftime or distance.

As used herein, pomalidomide refers to the following compound:

7.1 Compound I

In certain embodiments, Compound I for use in the methods providedherein, including the combination therapy, and in compositions providedherein is a compound of formula:

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer, racemic mixture, co-crystal, clathrate, or polymorph thereof

In one embodiment, the compound is3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione.

Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer, racemic mixture, co-crystal, clathrate, orpolymorph thereof can be prepared by methods known to one of skill inthe art, for example, according to the procedure described in U.S. Pat.No. 7,635,700 and U.S. Provisional App. No. 61/451,806.

In certain embodiments, the compound of Formula I is a solid. In certainembodiments, the compound of Formula I is hydrated. In certainembodiments, the compound of Formula I is solvated. In certainembodiments, the compound of Formula I is anhydrous. In certainembodiments, the compound of Formula I is nonhygroscopic.

In certain embodiments, the solid compound of Formula I is amorphous. Incertain embodiments, the solid compound of Formula I is crystalline. Incertain embodiments, the solid compound of Formula I is in a crystallineform described in U.S. Provisional Pat. App. No. 61/451,806, filed Mar.11, 2011, which is incorporated herein by reference in its entirety.

The solid forms of the compound of Formula I can be prepared accordingto the methods described in the disclosure of U.S. Provisional Pat. App.No. 61/451,806. The solid forms can be also prepared according to othermethods apparent to those of skill in the art.

In certain embodiments, the compound of Formula I is a hydrochloridesalt of3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable solvate, hydrate, co-crystal, clathrate, or polymorphthereof. In certain embodiments, the hydrochloride salt is a solid. Incertain embodiments, the hydrochloride salt is anhydrous. In certainembodiments, the hydrochloride salt is nonhygroscopic. In certainembodiments, the hydrochloride salt is amorphous. In certainembodiments, the hydrochloride salt is crystalline. In certainembodiments, the hydrochloride salt is in crystalline Form A.

The hydrochloride salt of the compound of Formula I and solid formsthereof can be prepared according to the methods described in thedisclosure of U.S. Provisional Pat. App. No. 61/451,806. Thehydrochloride salt the solid forms thereof can be also preparedaccording to other methods apparent to those of skill in the art.

The compound of Formula I provided herein contains one chiral center,and can exist as a mixture of enantiomers, e.g., a racemic mixture. Thisdisclosure encompasses the use of stereomerically pure forms of such acompound, as well as the use of mixtures of those forms. For example,mixtures comprising equal or unequal amounts of the enantiomers of thecompound of Formula I provided herein may be used in methods andcompositions disclosed herein. These isomers may be asymmetricallysynthesized or resolved using standard techniques such as chiral columnsor chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers,Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, S. H., Tablesof Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed.,Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of the structure.

7.2 Methods of Treatment

Provided herein are methods of treating, preventing, and/or managingdiseases, disorders and/or conditions associated immune-related andinflammatory diseases comprising administering a therapeuticallyeffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient in need thereof. In certain embodiments, the disease isselected from lupus, scleroderma, Sjögren syndrome, ANCA-inducedvasculitis, anti-phospholipid syndrome and myasthenia gravis. In certainembodiments, the disease is lupus or scleroderma.

The sensitivity of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof canbe studied in various in vivo and in vitro assays, including animalmodels known to one of skill in the art for immune-related andinflammatory diseases, including, but not limited to MRL/MpJ-Faslpr/Jmouse model of systemic lupus erythematosus, NZBWF1/J mouse model ofsystemic lupus erythematosus, bleomycin-induced skin fibrosis model, andmurine tight skin-1 (Tsk-1) mouse model.

7.2.1 Treatment of Scleroderma

In certain embodiments, provided herein are methods of treating,preventing, and/or managing scleroderma or a symptom thereof, comprisingadministering a therapeutically effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to a patient having scleroderma.

In certain embodiments, provided herein are methods of preventingscleroderma or a symptom thereof, comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to a patientat risk of having scleroderma.

In certain embodiments, the scleroderma is localized, systemic, limitedor diffuse scleroderma.

In certain embodiments, the systemic scleroderma comprises CRESTsyndrome (Calcinosis, Raynaud's syndrome, esophagaeal dysfunction ordysmotility, sclerodactyly, telangiectasia). Scleroderma is also knownas systemic sclerosis or progressive systemic sclerosis. In certainembodiments, provided herein are methods of treating or preventingRaynaud's disease or syndrome. In certain embodiments, systemicsclerosis comprises scleroderma lung disease, scleroderma renal crisis,cardiac manifestations, muscular weakness (including fatigue or limitedCREST), gastrointestinal dysmotility and spasm, and abnormalities in thecentral, peripheral and autonomic nervous system (including carpaltunnel syndrome followed by trigeminal neuralgia). It also includesgeneral disability, including depression, and impact on quality of life.

In certain embodiments, limited scleroderma is limited to the hands, theface, neck, or combinations thereof.

In certain embodiments, diffuse scleroderma comprises skin tighteningand also occurs above the wrists (or elbows). In certain embodiments,the diffuse systemic sclerosis is sine scleroderma, comprising internalorgan fibrosis, but no skin tightening; or familial progressive systemicsclerosis.

In one embodiment, scleroderma is not associated with wasting, such asdisease-related wasting.

In one embodiment, provided herein are methods for the reduction,inhibition, or prevention of one or more of the following symptoms ofscleroderma: (i) gradual hardening, thickening, and tightening of theskin (e.g., in extremities, such as hands, face, and feet); (ii) skindiscoloration; (iii) numbness of extremities; (iv) shiny skin; (v) smallwhite lumps under the surface of the skin that erupt into a chalky whitefluid; (vi) Raynaud's esophagaeal dysfunction (pain, numbness, and/orcolor changes in the hands caused by spasm of the blood vessels uponexposure to cold or emotional stress); (vii) telangiectasia (red spotson, e.g., the hands, palms, forearms, face, and lips); (viii) painand/or stiffness of the joints; (ix) swelling of the hands and feet; (x)itching of the skin; (xi) stiffening and curling of the fingers; (xii)ulcers (sores) on the outside of certain joints, such as knuckles andelbows; (xiii) digestive problems, such as heartburn, difficulty inswallowing, diarrhea, irritable bowel, and constipation; (xiv) fatigueand weakness; (xv) shortness of breath; (xvi) arthritis; (xvii) hairloss; (xviii) internal organ problems; (xix) digital ulcers; or (xx)digital auto-amputation, comprising administering an effective amount ofCompound I to a patient in need thereof.

Without being bound to any particular theory, it is believed thatCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof enhances Th1 immuneresponse, and suppresses Th2 immune response, which may result inanti-fibrotic effects in the skin.

Further provided herein are methods for improving or reducing the skinthickness of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the skin thickness is reduced by about20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%about 80%, about 90% or more.

Further provided herein are methods for achieving one or more clinicalendpoints associated with scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient in need thereof.

Further provided herein are methods for increasing the overall survival,objective response rate, time to progression, progression-free survivaland/or time-to-treatment failure of a patient having sclerodermacomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for decreasing mortality,respiratory mortality and/or respiratory hospitalization of a patienthaving scleroderma comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for improving the modified Rodnanskin score of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the improvement in modified Rodnan skinscore is 5, 10, 15 or 20 points or more.

Further provided herein are methods for improving or reducing the skinthickness of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the skin thickness is reduced by about20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%about 80%, about 90% or more.

Further provided herein are methods for improving or reducing skininduration of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient.

Further provided herein are methods for improving the dermatologyquality of life index of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

Further provided herein are methods for improving the pulmonary functionof a patient having scleroderma comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to thepatient.

Further provided herein are methods for improving the carbon monoxidediffusing capacity of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the carbon monoxidediffusing capacity of a patient is improved by an improvement in thediffusing capacity of the lung for carbon monoxide (D_(L)co) of about10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%,about 70% about 80%, about 90% or more.

Further provided herein are methods for improving the Mahler Dyspneaindex of a patient having scleroderma comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof tothe patient. In one embodiment, the improvement in Mahler Dyspnea indexis 4, 5, 6, 7, 8, 9 or 10 points or more.

Further provided herein are methods for improving the Saint George'sRespiratory Questionnaire score of a patient having sclerodermacomprising administering an effective amount of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to the patient. In one embodiment,the improvement in Saint George's Respiratory Questionnaire score is 4,8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 points or more.

Further provided herein are methods for improving the UCLA sclerodermaclinical trial consortium gastrointestinal tract score of a patienthaving scleroderma comprising administering an effective amount ofCompound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof to the patient.

Further provided herein are methods for treating or preventing digitalulcer of a patient or patient population having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

Further provided herein are methods improving flow-mediated dilatationof a patient having scleroderma comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to thepatient.

Further provided herein are methods improving or increasing the sixminute walk distance of a patient having scleroderma comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient. In one embodiment, the improvement inthe six minute walk distance is about 200 meters, about 250 meters,about 300 meters, about 350 meters, about 400 meters or more.

7.2.2 Treatment of Lupus Erythematosus

In certain embodiments, provided herein are methods of treating,preventing, and/or managing lupus erythematosus or a symptom thereof,comprising administering a therapeutically effective amount of CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof to a patient having lupuserythematosus.

In one embodiment, provided herein are methods of preventing lupuserythematosus or a symptom thereof, comprising administering aneffective amount of Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof toa patient at risk of having lupus erythematosus.

In certain embodiments, provided herein are methods for treating,preventing, and/or managing systemic lupus erythematosus (SLE),cutaneous lupus erythematosus (CLE) or drug-induced lupus.

The phrase “Systemic lupus erythematosus” is interchangeably used hereinwith SLE and lupus and refers to all manifestations of the disease asknown in the art (including remissions and flares). In SLE, abnormalhyperactivity of B lymphocytes and massive abnormal production ofimmunoglobulin gamma (IgG) auto-antibodies play a key role. Thispathological process results in sequestration and destruction ofIg-coated cells, fixation and cleaving of complement proteins, andrelease of chemotaxins, vasoactive peptides and destructive enzymes intotissues (Hahn B H. Systemic Lupus Erythematosus. In: Kasper D L,Braunwald E, Fauci A S, Hauser S L, Longo D L, Jameson, J L, editors.In: Harrison's Principles of Internal Medicine (16th edition). New York(US): McGraw-Hill; 2005. pp. 1960-1967).

Symptoms of SLE vary from person to person, and may come and go. In mostpatients, the symptoms include joint pain and swelling. Frequentlyaffected joints are the fingers, hands, wrists, and knees. Some patientsdevelop arthritis. Other common symptoms include: chest pain when takinga deep breath, fatigue, fever with no other cause, general discomfort,uneasiness, or ill feeling (malaise), hair loss, mouth sores, swollenlymph nodes, sensitivity to sunlight, skin rash-a “butterfly” rash overthe cheeks and bridge of the nose affects about half of people with SLE,in some patients, the rash gets worse in sunlight, and the rash may alsobe widespread.

Other symptoms depend on what part of the body is affected, and mayinclude the following:

Brain and nervous system: headaches, numbness, tingling, seizures,vision problems, personality changes,

Digestive tract: abdominal pain, nausea, and vomiting,

Heart: abnormal heart rhythms (arrhythmias),

Lung: coughing up blood and difficulty breathing, and

Skin: patchy skin color, fingers that change color when cold (Raynaud'sphenomenon).

Some patients only have skin symptoms. This is called discoid lupus.

In one embodiment, provided herein are methods of treating moderate,severe, or very severe SLE. The term “severe SLE” as used herein refersto an SLE condition where the patient has one or more severe orlife-threatening symptoms (such as hemolytic anemia, extensive heart orlung involvement, kidney disease, or central nervous systeminvolvement).

Further provided herein are methods for achieving one or more clinicalendpoints associated with SLE comprising administering an effectiveamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to a patientin need thereof.

Further provided herein are methods for increasing the overall survival,objective response rate, time to progression, progression-free survivaland/or time-to-treatment failure of a patient having SLE comprisingadministering an effective amount of Compound I or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, tautomer or racemicmixtures thereof to the patient.

In certain embodiment, Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereofacts as an inhibitor of primary human memory CD19+B-cell differentiationto the plasmablast stage. Without being bound to any particular theory,it is believed that Compound I or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereofblocks cells at a premature stage thereby decreasing the numbers ofplasmablasts that are capable of producing high levels ofimmunoglobulin. A functional consequence of this effect is reducedimmunoglobulin G (IgG) and immunoglobulin M (IgM) production in thesedifferentiation cultures.

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof inhibits of the ability of primary human memory CD19+B-cells todifferentiate to the plasmablast stage. In certain embodiments, CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof has no significant effect on matureCD138+ plasma cells in short term cultures. In certain embodiments,Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof inhibits B celldifferentiation factors including interferon regulatory factor 4 (IRF4),lymphocyte-induced maturation protein (BLIMP), X-box-protein-1 (XBP-1)and B cell lymphoma 6 (Bc16).

7.2.3 Treatment of Other Immune-Related Diseases or Disorders

Further provided herein are methods of treating, managing, or preventingother immune-related diseases or conditions using Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof. In certain embodiments, forexample, provided herein is a method of treating an individual having adisease or disorder, wherein the disease or disorder is caused by, or isassociated with, an inappropriate or undesirable immune response, e.g.,a disease, disorder or condition that can be treated beneficially byimmunosuppression, comprising administering to the individual Compound Ior a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof.

In various specific embodiments, said immune-related disease is one ormore of selected from Sjögren syndrome, ANCA-induced vasculitis,anti-phospholipid syndrome, myasthenia gravis, Addison's disease,alopecia areata, ankylosing spondylitis, antiphospholipid antibodysyndrome, antiphospholipid syndrome (primary or secondary), asthma,autoimmune gastritis, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease, autoimmune lymphoproliferative disease,autoimmune thrombocytopenic purpura, Balo disease, Behcet's disease,bullous pemphigoid, cardiomyopathy, celiac disease, Chagas disease,chronic inflammatory demyelinating polyneuropathy, cicatrical pemphigoid(e.g., mucous membrane pemphigoid), cold agglutinin disease, degosdisease, dermatitis hepatiformis, essential mixed cryoglobulinemia,Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome,Hashimoto's thyroiditis (Hashimoto's disease; autoimmune thyroditis),idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura, IgAnephropathy, juvenile arthritis, lichen planus, Ménière disease, mixedconnective tissue disease, morephea, narcolepsy, neuromyotonia,pediatric autoimmune neuropsychiatric disorders (PANDAs), pemphigusvulgaris, pernicious anemia, polyarteritis nodosa, polychondritis,polymyalgia rheumatica, primary agammaglobulinemia, primary biliarycirrhosis, Raynaud disease (Raynaud phenomenon), Reiter's syndrome,relapsing polychondritis, rheumatic fever, Sjogren's syndrome,stiff-person syndrome (Moersch-Woltmann syndrome), Takayasu's arteritis,temporal arteritis (giant cell arteritis), uveitis, vasculitis (e.g.,vasculitis not associated with lupus erythematosus), vitiligo, and/orWegener's granulomatosis.

7.3 Dosages and Dosing Amounts

The dose of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof to beadministered to a patient is rather widely variable and can be subjectto the judgment of a health-care practitioner. Doses of Compound I or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof vary depending on factors such as:specific indication to be treated, prevented, or managed; age andcondition of a patient; and amount of second active agent used, if any.In general, Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof can beadministered one to four or more times a day in a dose of about 0.005mg/kg of a patient's body weight to about 10 mg/kg of a patient's bodyweight in a patient, but the above dosage may be properly varieddepending on the age, body weight and medical condition of the patientand the type of administration. In one embodiment, the dose is about0.01 mg/kg of a patient's body weight to about 5 mg/kg of a patient'sbody weight, about 0.05 mg/kg of a patient's body weight to about 1mg/kg of a patient's body weight, about 0.1 mg/kg of a patient's bodyweight to about 0.75 mg/kg of a patient's body weight or about 0.25mg/kg of a patient's body weight to about 0.5 mg/kg of a patient's bodyweight.

In one embodiment, one dose is given per day. In any given case, theamount of Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof administeredwill depend on such factors as the solubility of the active component,the formulation used and the route of administration. In one embodiment,application of a topical concentration provides intracellular exposuresor concentrations of about 0.01-10 μM.

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof is used in an amount of from about 0.1 mg to about 1000 mg perday, and can be adjusted in a conventional fashion (e.g., the sameamount administered each day of the treatment, prevention or managementperiod), in cycles (e.g., one week on, one week off), or in an amountthat increases or decreases over the course of treatment, prevention, ormanagement. In other embodiments, the dose can be from about 1 mg toabout 300 mg, from about 0.1 mg to about 150 mg, from about 1 mg toabout 200 mg, from about 10 mg to about 100 mg, from about 0.1 mg toabout 50 mg, from about 1 mg to about 50 mg, from about 10 mg to about50 mg, from about 20 mg to about 30 mg, or from about 1 mg to about 20mg.

7.4 Combination Therapy

Compound I or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, tautomer or racemic mixtures thereof can be combined withother pharmacologically active compounds (“second active agents”) inmethods and compositions provided herein. Certain combinations may worksynergistically in the treatment of particular types diseases ordisorders, and conditions and symptoms associated with such diseases ordisorders. Compound I or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, tautomer or racemic mixtures thereof can alsowork to alleviate adverse effects associated with certain second activeagents, and vice versa.

One or more second active ingredients or agents can be used in themethods and compositions provided herein. Second active agents can belarge molecules (e.g., proteins) or small molecules (e.g., syntheticinorganic, organometallic, or organic molecules).

In another embodiment, the method of treatment provided herein comprisesthe administration of a second therapeutic agent, wherein the secondtherapeutic agent is an anti-inflammatory drug, e.g., a steroidalanti-inflammatory drug, or a non-steroidal anti-inflammatory drug(NSAID), acetaminophen, naproxen, ibuprofen, acetylsalicylic acid, andthe like. In a more specific embodiment in which an NSAID isadministered, a proton pump inhibitor (PPI), e.g., omeprazole may alsoadministered. In one embodiment, the antiinflammatory agent is acorticosteroid. In another embodiment, the antiinflammatory agent iscolchicine.

In another embodiment, the second therapeutic agent is animmunomodulatory compound or an immunosuppressant compound such asazathioprine (Imuran™, Azasan™), methotrexate (Rheumatrex™, Trexall™),penicillamine (Depen™, Cuprimine™), cyclophosphamide (Cytoxan™)mycophenalate (CellCept™, Myfortic™), bosentan (Tracleer®), prednisone(Deltasone™, Liquid Pred™), and a PDE5 inhibitor. In another embodiment,where the affected individual has digital ulcerations and pulmonaryhypertension, a vasodilator such as prostacyclin (iloprost) may beadministered.

In another embodiment, the second therapeutic agent is an inhibitor ofActRII receptors or an activin-ActRII inhibitor. Inhibitors of ActRIIreceptors include ActRIIA inhibitors and ActRIIB inhibitors. Inhibitorsof ActRII receptors can be polypeptides comprising activin-bindingdomains of ActRII. In certain embodiments, the activin-binding domaincomprising polypeptides are linked to an Fc portion of an antibody(i.e., a conjugate comprising an activin-binding domain comprisingpolypeptide of an ActRII receptor and an Fc portion of an antibody isgenerated). In certain embodiments, the activin-binding domain is linkedto an Fc portion of an antibody via a linker, e.g., a peptide linker.

An exemplary activin-binding ActRIIA polypeptide fused to a human Fcdomain is provided in SEQ ID NO: 1.

SEQ ID NO: 1 ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An exemplary fusion protein comprising a soluble extracellular domain ofActRIIB fused to an Fc domain is provided in SEQ ID NO: 2.

SEQ ID NO: 2 ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Further examples of non-antibody proteins selected for activin orActRIIA binding and methods for design and selection of the same arefound in WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989,US 2003/0133939, and US 2005/0238646, each of which is incorporatedherein by reference in its entirety.

Any combination of the above therapeutic agents, suitable for treatmentof the diseases or symptoms thereof, can be administered. Suchtherapeutic agents can be administered in any combination with CompoundI or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixtures thereof, at the same time or as a separatecourse of treatment.

7.5 Cycling Therapy

In certain embodiments, Compound I or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, tautomer or racemic mixturesthereof provided herein is cyclically administered to a patient. Cyclingtherapy involves the administration of an active agent for a period oftime, followed by a rest (i.e., discontinuation of the administration)for a period of time, and repeating this sequential administration.Cycling therapy can reduce the development of resistance to one or moreof the therapies, avoid or reduce the side effects of one of thetherapies, and/or improve the efficacy of the treatment.

Consequently, in one embodiment, a compound provided herein isadministered daily in a single or divided doses in a four to six weekcycle with a rest period of about a week or two weeks. Cycling therapyfurther allows the frequency, number, and length of dosing cycles to beincreased. Thus, another embodiment encompasses the administration of acompound provided herein for more cycles than are typical when it isadministered alone. In yet another embodiment, a compound providedherein is administered for a greater number of cycles than wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, a compound provided herein is administered daily andcontinuously for three or four weeks at a dose of from about 0.1 mg toabout 500 mg per day, followed by a rest of one or two weeks. In otherembodiments, the dose can be from about 1 mg to about 300 mg, from about0.1 mg to about 150 mg, from about 1 mg to about 200 mg, from about 10mg to about 100 mg, from about 0.1 mg to about 50 mg, from about 1 mg toabout 50 mg, from about 10 mg to about 50 mg, from about 20 mg to about30 mg, or from about 1 mg to about 20 mg, followed by a rest.

In one embodiment, a compound provided herein and a second activeingredient are administered orally, with administration of the compoundprovided herein occurring 30 to 60 minutes prior to the second activeingredient, during a cycle of four to six weeks. In another embodiment,the combination of a compound provided herein and a second activeingredient is administered by intravenous infusion over about 90 minutesevery cycle.

Typically, the number of cycles during which the combination treatmentis administered to a patient will be from about one to about 24 cycles,from about two to about 16 cycles, or from about four to about threecycles.

7.6 Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms provided herein comprise a compound provided herein, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,racemate, clathrate, or prodrug thereof. Pharmaceutical compositions anddosage forms can further comprise one or more excipients.

Pharmaceutical compositions and dosage forms provided herein can alsocomprise one or more additional active ingredients. Examples of optionalsecond, or additional, active ingredients are disclosed above.

Single unit dosage forms provided herein are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), topical (e.g., eye drops or other ophthalmicpreparations), transdermal or transcutaneous administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; eye drops or other ophthalmic preparations suitable fortopical administration; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms will typically varydepending on their use. For example, a dosage form used in the acutetreatment of a disease may contain larger amounts of one or more of theactive ingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Theseand other ways in which specific dosage forms are used will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 20^(th) ed., Mack Publishing,Easton Pa. (2000).

In one embodiment, pharmaceutical compositions and dosage forms compriseone or more excipients. Suitable excipients are well known to thoseskilled in the art of pharmacy, and non-limiting examples of suitableexcipients are provided herein. Whether a particular excipient issuitable for incorporation into a pharmaceutical composition or dosageform depends on a variety of factors well known in the art including,but not limited to, the way in which the dosage form will beadministered to a patient. For example, oral dosage forms such astablets may contain excipients not suited for use in parenteral dosageforms. The suitability of a particular excipient may also depend on thespecific active ingredients in the dosage form. For example, thedecomposition of some active ingredients may be accelerated by someexcipients such as lactose, or when exposed to water. Active ingredientsthat comprise primary or secondary amines are particularly susceptibleto such accelerated decomposition. Consequently, provided arepharmaceutical compositions and dosage forms that contain little, ifany, lactose other mono- or di-saccharides. As used herein, the term“lactose-free” means that the amount of lactose present, if any, isinsufficient to substantially increase the degradation rate of an activeingredient.

Lactose-free compositions can comprise excipients that are well known inthe art and are listed, for example, in the U.S. Pharmacopeia (USP)25-NF20 (2002). In general, lactose-free compositions comprise activeingredients, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. In one embodiment,lactose-free dosage forms comprise active ingredients, microcrystallinecellulose, pre-gelatinized starch, and magnesium stearate.

Also provided are anhydrous pharmaceutical compositions and dosage formscomprising active ingredients, since water can facilitate thedegradation of some compounds. For example, the addition of water (e.g.,5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long-term storage in order to determine characteristics suchas shelf-life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms can be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions. Pharmaceutical compositions and dosage formsthat comprise lactose and at least one active ingredient that comprisesa primary or secondary amine are anhydrous if substantial contact withmoisture and/or humidity during manufacturing, packaging, and/or storageis expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are, in one embodiment, packaged using materials known toprevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastics, unit dose containers(e.g., vials), blister packs, and strip packs.

Also provided are pharmaceutical compositions and dosage forms thatcomprise one or more compounds that reduce the rate by which an activeingredient will decompose. Such compounds, which are referred to hereinas “stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. In one embodiment, dosage forms comprise a compoundprovided herein in an amount of from about 0.10 to about 500 mg. Inother embodiments, dosage forms comprise a compound provided herein inan amount of about 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50,100, 150, 200, 250, 300, 350, 400, 450, or 500 mg.

In other embodiments, dosage forms comprise the second active ingredientin an amount of 1 to about 1000 mg, from about 5 to about 500 mg, fromabout 10 to about 350 mg, or from about 50 to about 200 mg. Of course,the specific amount of the second active agent will depend on thespecific agent used, the diseases or disorders being treated or managed,and the amount(s) of a compound provided herein, and any optionaladditional active agents concurrently administered to the patient.

Oral Dosage Forms

Pharmaceutical compositions that are suitable for oral administrationcan be provided as discrete dosage forms, such as, but not limited to,tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g.,flavored syrups). Such dosage forms contain predetermined amounts ofactive ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Oral dosage forms provided herein are prepared by combining the activeingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

In one embodiment, oral dosage forms are tablets or capsules, in whichcase solid excipients are employed. In another embodiment, tablets canbe coated by standard aqueous or nonaqueous techniques. Such dosageforms can be prepared by any of the methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose,pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions is, in oneembodiment, present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants may be used in the compositions to provide tablets thatdisintegrate when exposed to an aqueous environment. Tablets thatcontain too much disintegrant may disintegrate in storage, while thosethat contain too little may not disintegrate at a desired rate or underthe desired conditions. Thus, a sufficient amount of disintegrant thatis neither too much nor too little to detrimentally alter the release ofthe active ingredients may be used to form solid oral dosage forms. Theamount of disintegrant used varies based upon the type of formulation,and is readily discernible to those of ordinary skill in the art. In oneembodiment, pharmaceutical compositions comprise from about 0.5 to about15 weight percent of disintegrant, or from about 1 to about 5 weightpercent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, other starches,clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms include, but are not limited to, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zincstearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.Additional lubricants include, for example, a syloid silica gel(AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), acoagulated aerosol of synthetic silica (marketed by Degussa Co. ofPlano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants may be used in an amount of less than about 1 weight percentof the pharmaceutical compositions or dosage forms into which they areincorporated.

In one embodiment, a solid oral dosage form comprises a compoundprovided herein, anhydrous lactose, microcrystalline cellulose,polyvinylpyrrolidone, stearic acid, colloidal anhydrous silica, andgelatin.

Controlled Release Dosage Forms

Active ingredients such as the compounds provided herein can beadministered by controlled release means or by delivery devices that arewell known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U.S. Pat. Nos. 3,845,770;3,916,899; 3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595;5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480;5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945;5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363;6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358;6,699,500 each of which is incorporated herein by reference. Such dosageforms can be used to provide slow or controlled release of one or moreactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled release formulations known to those ofordinary skill in the art, including those described herein, can bereadily selected for use with the active ingredients provided herein.Thus, the compositions provided encompass single unit dosage formssuitable for oral administration such as, but not limited to, tablets,capsules, gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased subject compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the drug may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In one embodiment, a pump may be used(see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used. In yetanother embodiment, a controlled release system can be placed in asubject at an appropriate site determined by a practitioner of skill,i.e., thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138(1984)). Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)). The active ingredient can bedispersed in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The active ingredient then diffuses through the outerpolymeric membrane in a release rate controlling step. The percentage ofactive ingredient in such parenteral compositions is highly dependent onthe specific nature thereof, as well as the needs of the subject.

Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. In someembodiments, administration of a parenteral dosage form bypassespatients' natural defenses against contaminants, and thus, in theseembodiments, parenteral dosage forms are sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, but arenot limited to: Water for Injection USP; aqueous vehicles such as, butnot limited to, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, and Lactated Ringer'sInjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and polypropylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms. For example, cyclodextrin and its derivativescan be used to increase the solubility of a compound provided herein.See, e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms provided herein include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th), 18^(th) and 20^(th) eds., Mack Publishing, Easton Pa. (1980,1990 and 2000); and Introduction to Pharmaceutical Dosage Forms, 4thed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedherein are well known to those skilled in the pharmaceutical arts, anddepend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied. In one embodiment,excipients include, but are not limited to, water, acetone, ethanol,ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,isopropyl palmitate, mineral oil, and mixtures thereof to formsolutions, emulsions or gels, which are non-toxic and pharmaceuticallyacceptable. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms. Examples of additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th), 18^(th) and 20^(th) eds., MackPublishing, Easton Pa. (1980, 1990 and 2000).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients. Also,the polarity of a solvent carrier, its ionic strength, or tonicity canbe adjusted to improve delivery. Compounds such as stearates can also beadded to pharmaceutical compositions or dosage forms to alter thehydrophilicity or lipophilicity of one or more active ingredients so asto improve delivery. In other embodiments, stearates can serve as alipid vehicle for the formulation, as an emulsifying agent orsurfactant, or as a delivery-enhancing or penetration-enhancing agent.In other embodiments, salts, solvates, hydrates, prodrugs, clathrates,or stereoisomers of the active ingredients can be used to further adjustthe properties of the resulting composition.

Kits

In one embodiment, active ingredients provided herein are notadministered to a patient at the same time or by the same route ofadministration. In another embodiment, provided are kits which cansimplify the administration of appropriate amounts of activeingredients.

In one embodiment, a kit comprises a dosage form of a compound providedherein. Kits can further comprise additional active ingredients such asother anti-inflammatory, immunomodulatory or immunosuppressantcompounds, or a combination thereof. Examples of the additional activeingredients include, but are not limited to, those disclosed herein.

In other embodiments, kits can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits can further comprise cells or blood for transplantation as well aspharmaceutically acceptable vehicles that can be used to administer oneor more active ingredients. For example, if an active ingredient isprovided in a solid form that must be reconstituted for parenteraladministration, the kit can comprise a sealed container of a suitablevehicle in which the active ingredient can be dissolved to form aparticulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

8. EXAMPLES

The following Examples are presented by way of illustration, notlimitation.

8.1 Example 1 Effect of Test Compound on Cytokine and ChemokineProduction in Anti-Human CD3-Stimulated Human T Cells

This example demonstrates the effect of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione (testcompound) on cytokine and chemokine production in anti-humanCD3-stimulated human T cells using multiplex Luminex Technology.

The following abbreviations are used:

Abbreviation Explanation or Definition IL Interleukin G-CSF GranulocyteColony Stimulating Factor GM-CSF Granulocyte Macrophage ColonyStimulating Factor IFN-γ Interferon Gamma TNF-α Tumor Necrosis FactorAlpha RANTES Regulated on Activation, Normal T Cell Expressed andSecreted

The following materials were used in this study:

RPMI-1640 Media supplemented with 10% FBS, 100 units/mL penicillin, 100mg/mL streptomycin and 2 mM L-glutamine (Life Technologies)

RosetteSep® Human T—Cell Enrichment Cocktail (StemCell, Cat#15061)

Luminex Human Cytokine/Chemokine 12-Plex Kit (Millipore, Cat#MPXHCYTO-60K-12)

Luminex IS100 instrument (Millipore)

Anti-human CD3 antibody, OKT3 clone (eBioscience, Cat#16-0037-85)

The test compounds were prepared as stock solutions of 4 mM in DMSO. Tcells were isolated from buffy coat by negative selection using theRosetteSep® T Cell Enrichment Cocktail according to manufacturer'sprocedures.

All 96-well plates were pre-coated with 3 μg/mL anti-human CD3 antibodyin 100 μL 1×PBS for 4 hours at 37° C. The plates were washed 3 timeswith RPMI-1640 Complete Media prior to the T cell assay. The T cellswere then plated in anti-CD3-pre-coated plates at a density of 2.5×10⁵cells/well in 180 μL RPMI-1640 Complete Media. The cells were treatedwith 20 μL 10× titrated test compound at 10, 1, 0.1, 0.01, 0.001,0.0001, and 0.00001 μM in duplicate. The final DMSO concentrations were0.25%. The plates were incubated for 48 hours at 37° C., 5% CO₂.

After 48 hours, the supernatants were harvested and tested by amultiplex cytometric bead array (CBA) assay for the followingcytokines/chemokines: IL-2, IL-3, IL-5, IL-10, IL-13, IL-15, IL-17A,GM-CSF, G-CSF, IFN-γ, TNF-α, and RANTES. The CBA plates were analyzed onthe Luminex IS100 instrument.

Data from each donor was graphed using GraphPad Prism 5.0 software andexpressed as mean pg/mL±SEM and % of DMSO control±SEM.

The test compound demonstrated immunomodulatory activity in anti-CD3stimulated primary human T cells, altering the production of severalcytokines and chemokines. Baseline levels of cytokines and chemokinesproduced by stimulated human T cells incubated with vehicle arepresented in Table 1 below.

TABLE 1 Baseline levels of cytokines and chemokines Baseline AmountProduced Cytokine/Chemokine (pg/mL) IL-2 31 IL- 38 IL-5 27 IL-10 449IL-13 205 IL-17A 19 GM-CSF 132 IFN-γ 1271 TNF-α 411 RANTES 314

The test compound enhanced IL-2, IL-3, IL-5, IL-10, IL-13, GM-CSF,IFN-γ, RANTES, and TNF-α production in stimulated human T cells. Theenhancement of production by the test compound was largelyconcentration-dependent for most of the cytokines and chemokines, exceptfor IL-10 and IL-5. The test compound enhanced IL-10 production at lowerconcentrations but inhibited enhancement of IL-10 production at higherconcentrations. The test compound increased IL-5 production 3- and4-fold at 0.01 and 0.1 μM, respectively, showing less enhancement atboth lower and higher concentrations. The effect of the test compound oncytokine and chemokine production in anti-CD3-stimulated human T cells,expressed as absolute amount produced and as percentage of vehiclecontrol cells are provided in FIGS. 1 and 2, respectively. The dashedline denotes the level equivalent to double the baseline production(EC₂₀₀) in FIG. 2.

8.2 Example 2 Anti-Inflammatory Activity

Anti-inflammatory activity of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione (testcompound) was studied in human peripheral blood mononuclear cells(hPBMC). Luminex Technology was used to determine the inhibitory(enhancement) concentration, IC₅₀ for the compound for the simultaneousprofiling of pro-inflammatory cytokines/chemokines and IL-10(anti-inflammatory cytokine) from LPS-stimulated healthy human donorPBMCs.

The following abbreviations are used:

Abbreviation Explanation or Definition GM-CSF Granulocyte MacrophageColony Stimulating Factor IL Interleukin LPS lipopolysaccharide MCP-1monocyte chemotactic protein-1 MDC Macrophage-derived chemokine MIP-1αMacrophage inflammatory protein-1alpha MIP-1β Macrophage inflammatoryprotein-1beta PBMC Peripheral Blood Mononuclear cells PPM PleiotrophicPathway Modifier RANTES Regulated upon Activation Normal T-cellExpressed, and Secreted TNF-α Tumor Necrosis Factor-Alpha

50 ml Buffy coat from healthy donors was obtained from Blood Center ofNew Jersey (East Orange, N.J.). Lipopolysaccharide (strain)(Cat# L-1887)was purchased from Sigma. Milliplex kits with antibody bound beads forLuminex xMAP Technology was purchased from Millipore (Billerica, Mass.)and combined into multiplex format prior to assay.

Purification of Human Peripheral Blood Mononuclear Cells

50 ml human buffy coat was aliquoted 25 ml each into two 50 ml conicaltubes and 25 ml sterile HBSS was added to each conical tube. The tubeswere gently mixed by inverting. Fifteen ml of room temperatureFicoll-Paque Plus (GE Healthcare (location); cat#17-1440-02) wasaliquoted into four 50 ml conical tubes. Then 25 ml of the Buffycoat/HBSS mixture was layered gently and slowly on top of the Ficoll.The samples were centrifuged at 450 rpm for 35 minutes. The top layeredcontaining plasma was pipetted off and discarded. The interfacecontaining mononuclear cells was transferred into two 50 ml conicaltubes. Both conical tubes were filled to total volume of 50 ml with HBSSand centrifuged at 1200 rpm for 10 minutes. The cells were washed againin HBSS and spun at 1000 rpm for 10 minutes. Cell pellet was resuspendedwith 20 ml RPMI complete medium (RPMI/5% human sera/1× pen/strep/glut)and counted.

Treatment of Human Peripheral Blood Mononuclear Cells

One hundred μl (2×106/ml) of hPBMCs were added to each well of a 96 wellflat-bottom plate (final cell count=2×105/well) and incubated at 37° C.for 1 hour. Twenty μl (10×) compound was added to each test well andtwenty μl medium containing 2.5% DMSO was added to each control well([DMSO]final=0.25%) and plate was incubated for 1 hour at 37° C. Cellswere then stimulated with 80 μl of 2.5 ng/ml LPS ([LPS]final=1 ng/ml)and incubated for 18 hours at 37° C.

50 μl supernatant from each well was transferred into 3 newround-bottomed 96 well plates and stored at −20° C. for Luminexanalysis. Duplicate wells were performed for each sample.

Luminex Analysis

Supernatant samples were analyzed for cytokines in multiplex formataccording to the manufacturer's instructions (Millipore, Billerica, Ma01821) using a Luminex IS100 instrument. IL-12 and GM-CSF analyses weredone in a two-plex format using neat supernatants while all othercytokines were done in a multiplex format using supernatants diluted1:20. Data analysis was performed using Upstate Beadview software. IC₅₀swere calculated using non-linear regression, sigmoidal dose-response,constraining the top to 100% and bottom to 0%, allowing variable slope.The EC₅₀s were based on the upper constraint of the sigmoidal curvesequaling 246.9%, representing the average IL-10 enhancement produced bypomalidomide (control) at 10 μM

and the lower constraint to 100%. The IC₅₀ were performed using GraphPadPrism v5.00. The data values represent the mean+SEM (standard error ofthe mean) of n (number of experiments in duplicate).

As demonstrated by data in Table 2 below and FIG. 3, the test compoundhas varied potencies for the inhibitions of the multiple cytokinesexamined, e.g., Il-6, IL-8, IL-1β, GM-CSF, MDC, MIP-1α, MIP-1β, andTNF-α, in general. Also, the test compound enhanced production of IL-10,MCP-1, and RANTES with various potencies as provided in Table 3 and FIG.4.

TABLE 2 Summary of Cytokine Inhibitory Profile of Test Compound Testcompound Cytokine IC₅₀ (μM) IL-6 0.060 IL-8 >10 IL-1β 0.054 GM-CSF 0.95MDC 0.062 MIP-1α 0.30 MIP-1β >10 TNF-α 0.034

TABLE 3 Cytokine Profile Summary of-mean % of control at 0.1 μM Testcompound Cytokine (% of control) IL-10 480 MCP-1 236 RANTES 131

8.3 Example 3 Effect on Human Natural Killer (NK) Cell Function inResponse to IgG/Rituximab

In this example, the capacity of the test compound to enhance human NKcell function in response to IgG/Rituximab was studied. Theimmunomodulatory activity of the test compound was compared in twoassays of natural killer (NK) cell functions (1) IgG- and IL-2-inducedinterferon-gamma (IFN-γ) production and (2) killing activity, asmeasured in an in vitro ADCC (antibody-dependent cellular cytotoxity)model.

The following abbreviations are used:

Abbreviation Explanation or Definition ABC-DLBCL Activated B Cell-likeDiffuse Large B Cell Lymphoma ADCC Antibody Dependent CellularCytoxicity DMSO Dimethyl sulfoxide IgG Immunoglobulin G IFN-γInterferon-gamma NK Natural killer PPM Pleiotropic Pathway ModifierrhIL-2 Human Recombinant Interleukin-2

The materials used in the study and their sources are provided below:

Buffy Coat from healthy volunteers (Blood Center of New Jersey)

Ficoll-Hypaque Plus (Fisher Scientific Co LLC, PA, Cat #17144002)

RPMI-1640 Medium supplemented with 10% FBS (fetal bovine serum), 100units/mL penicillin,

100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen, Cat#21870-076)

RPMI-1640 Medium (without phenol red) supplemented with 10% FBS, 100units/mL penicillin,

100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen, Cat#11835-030)

Rituximab (Rituxan, Roche, Inc.) (Cat No. DIN 02241927, Lot No. B50177)

Human AB+ serum (Gemini Bio Products, CA, Cat #100-512)

CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, WI, Cat #G1780)

RosetteSep Human NK Cell Enrichment Cocktail (Stem Cell Technologies,Vancouver, BC, Cat#15065)

Mouse anti-human CD56+ conjugated APC (BD Biosciences, CA, Cat #555518)

Human Immunoglobulin G from Serum (IgG) (Sigma, St. Louis, Mo.; Cat#12511-10MG)

Human Recombinant IL-2 (R&D Systems, MN, Cat #202-IL-050/CF)

Human IFN-gamma ELISA Kit (ThermoFisher, Cat # PIEHIFNG5)

The following cell lines were used:

Activated B cell-like—diffuse large B cell lymphoma (ABC-DLBCL): Rivacells (NCI, MD)

Germinal center B-cell-like—diffuse large B cell lymphoma (GCB-DLBCL):

WSU-DLCL2 (Celgene Signal, CA)

Farage (ATCC, VA)

Follicular lymphoma: DoHH2 (DSMZ, Germany)

Burkitt's lymphoma (BL): Raji (ATCC, VA).

NK cells from healthy donors were isolated from buffy coat blood bynegative selection using the RosetteSep NK cell enrichment cocktail(Stem Cell Technologies, Vancouver, BC) prior to Ficoll-Hypaque (FisherScientific Co LLC, PA) density gradient centrifugation following themanufacturers' instructions. CD56+NK cells were isolated to ˜85% purity,as determined by flow cytometry (BD Biosciences, CA).

NK IgG-Induced Interferon-Gamma (IFN-Gamma) Assay

Ninety-six-well flat-bottom plates were coated with 100 μg/mL of humanIgG (Sigma) overnight at 4° C. The next day, unbound IgG was washed awaywith cold 1×PBS. NK cells were then plated in the IgG-coated 96-wellplates at 2×105 cells per well in 180 μL RPMI-1640 Media and 10 ng/mL ofrhIL-2 (R & D Systems, MN) was added. The test compound was added in avolume of 20 μL DMSO. Final concentrations of the test compound were0.0001, 0.001, 0.01, 0.1, 1, or 10 μM. Final DMSO concentrations were0.25%. After 48 hours, the supernatants were harvested and analyzed byELISA for IFN-γ production.

Data used to determine the ability of the test compound to enhance NKcell IFN-γ production in response to immobilized IgG and rhIL-2stimulation was analyzed for each donor using GraphPad Prism v5.0software. The data are presented in two ways, (1) as the absolute amountif IFN-γ produced (pg/mL±SEM) and (2) as the percentage of the amount ofIFN-γ produced in the presence of 1 μM pomalidomide. The EC₅₀ is theconcentration of the test compound providing half-maximal IFN-γproduction, with maximal production defined as the amount of IFN-γproduced in the presence of 1 μM pomalidomide. EC₅₀ values werecalculated using non-linear regression, sigmoidaldose-responseconstraining the top to 100% and bottom to 0% allowing for a variableslope. EC₅₀ for the test compound was 0.0015 μM.

The test compound enhanced NK cell IFN-γ□ production in a dose dependentmanner in response to immobilized IgG and IL-2 stimulation. Results areprovided in FIG. 5 (expressed as pg/mL of IFN-γ produced), respectively.FIG. 6 provides results expressed as a percentage of increased IFN-γproduced relative to the IFN-γ produced in the presence of pomalidomideat 1 μM for the test compound. Each value plotted in FIGS. 5 and 6represents the mean of 12-14 determinations±SEM.

ADCC Assay

Purified NK cells (5×104) were seeded in 96-well U-bottom plates in 100μL of RPMI-1640 medium without phenol (Invitrogen)+2% human AB+ serum(Gemini Bio Products, CA) and treated with 10 ng/mL rhIL-2 and rituximab(5 μg/mL) plus different concentrations of the test compound at 0.01 to10 μM for 48 hours. Various lymphoma cell lines (GCB-DLBCL: WSU-DLCL2and Farage; Follicular lymphoma:DoHH2; ABC-DLBCL: Riva; Burkitt'slymphoma [BL]: Raji) were treated with 5 μg/mL rituximab for 30 minutesat 37° C. Unbound rituximab was washed off, target cells (5×103/100μL/well) were added to the pretreated effector cells (NK cells) at a10:1 ratio, and the two were co-incubated for 4 hours at 37° C. Controlconditions consisted of NK cells plus tumor cells treated with (1)medium alone, (2) rituximab only, or (3) IL-2 alone. Using an aliquot ofsupernatant (50 μL), NK cell cytotoxicity against tumor cells wasanalyzed using a standard lactate dehydrogenease (LDH) release assay tomeasure ADCC (CytoTox 96 Non-Radioactive Cytoxicity Assay, Promega, WI).Spontaneous release by target cells alone was <15% of the maximumrelease, as determined with target cells lysed in 1% Triton X-100. Theexperimental release was corrected by subtraction of the spontaneousrelease of effector cells at the corresponding dilution. The percentageof specific lysis was calculated according to the formula:

Percentage specific lysis=100×(experimental−effector spontaneous−targetspontaneous)/(target maximum−target spontaneous).

The test compound induced dose-dependent NK cell-mediated ADCC in allcell lines. Three experiments were conducted for each cell line andsamples from each of three donors were tested in each experiment. Dataare presented in FIG. 8, as mean of 9 determinations±SEM.

8.4 Example 4 Effect on the Expression of Transcription Factors inPrimary Human B Cell Differentiation Model

In this example, the effect of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione (testcompound) on the expression of transcription factors controlling plasmacell differentiation, and immunoglobulin production, using an in vitrohuman B-cell differentiation culture system.

The following abbreviations are used in this example:

Abbreviation or Specialist Term Explanation or Definition BCL6 B-celllymphoma 6 protein BLIMP-1 B-lymphocyte-induced maturation protein 1EtOH Ethanol FBS Fetal bovine serum DMSO Dimethyl sulfoxide IRF-4Interferon regulatory factor 4 MFI Mean fluorescence intensity PAX5Paired box protein Pax-5 SLE Systemic lupus erythematosus XBP-1 X-boxbinding protein 1

50 ml Buffy coat from healthy donors were obtained from Blood Center ofNew Jersey. SLE Lupus PBMC samples were obtained from Conversant Bio(Huntsville, Ala. 35806).

The following cell culture reagents were used in this study.

ITEM Source Iscoves Modified Dulbecco Invitrogen medium Fetal BovineSerum Lonza Human Insulin Sigma Human Transferrin Sigmapenicillin/streptomycin Lonza Recombinant Human IL-2 R & D SystemsRecombinant Human IL-6 R & D Systems Recombinant Human IL-10 R & DSystems Recombinant Human IL-15 R & D Systems CD40 Ligand/TNFSF5/ R & DSystems histidine-tagged polyHistidine mouse IgG1 R & D Systems antibodyODN 2006-Human TLR9 Invivogen ligand Human Interferon ALPHA A PBinterferon source

The following were used in flow cytometry analysis.

ITEM Source FITC anti-human CD19 IBD Pharmigen FITC anti-human CD20 IBDPharmigen PE anti-human CD27 IBD Pharmigen PE anti-human CD38 IBDPharmigen APC anti-human CD38 IBD Pharmigen FITC anti-mouse IgG1kIsotype IBD Pharmigen PE anti-mouse IgG1k Isotype IBD Pharmigen FITCanti-mouse IgG2bk Isotype IBD Pharmigen APC anti-mouse IgG1k Isotype IBDPharmigen Stain Buffer IBD Pharmigen

The following gene primers were used for RT-PCR:

ITEM Source AICDA gene expression assay Applied Biosystem BCL6 geneexpression assay Applied Biosystem GAPDH gene expression assay AppliedBiosystem IGJ gene expression assay Applied Biosystem IRF4 geneexpression assay Applied Biosystem PAX5 gene expression assay AppliedBiosystem PRDM1 gene expression assay Applied Biosystem XBP1 geneexpression assay Applied Biosystem Reverse Transcription Kit AppliedBiosystem Master Mix Applied Biosystem

8.4.1 Purification of hPBMCs

Fifty ml human buffy coat was aliquoted 25 ml each into two 50 mlconical tubes and 25 ml sterile HBSS was added to each conical tube. Thetubes were gently mixed by inverting. Fifteen ml of room temperatureFicoll-Paque Plus (GE Healthcare; cat#17-1440-02) was aliquoted intofour 50 ml conical tubes. Then 25 ml of the Buffy coat/HBSS mixture waslayered gently and slowly on top of the Ficoll. The samples werecentrifuged at 450 rpm for 35 minutes. The top layered containing plasmawas pipetted off and discarded. The interface containing mononuclearcells was transferred into two 50 ml conical tubes. Both conical tubeswere filled to total volume of 50 ml with HBSS and centrifuged at 1200rpm for 10 minutes. The cells were washed again in HBSS and spun at 1000rpm for 10 minutes. Cell pellet was resuspended with 20 mL of B cellmedia (Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin) and countedon the cell counter.

8.4.2 B Cell Enrichment CD19+

Purified PBMCs were counted and aliquoted at 2×10⁸ cells per tube. Thecells were centrifuged at 1200 rpm for 5 minutes and then supernatantswere discarded. The cells were resuspended in 4 mL of Robosep Buffer(Stemcell Technologies catalog #20104) and transferred to a 14 mLpolystyrene round bottom tube (BD catalog #352057) and mixed well. Then200 μL of EasySep Human B cell enrichment cocktail was added (StemCellTechnologies catalog #19054). Samples were vortexed and incubated atroom temperature for 10 minutes. Next 300 μL of EasySep Magneticparticles (vortexed) (StemCell Technologies catalog #19054) were addedto the tube. Samples were vortexed and incubated at room temperature for5 minutes. After the 5 minute incubation, 5 mL of Robosep buffer wasadded to the tube and mixed well by pipetting up and down. The tube wasimmediately places in the silver magnet (StemCell Technologies catalog#19054) and incubated at room temperature for 5 minutes. Afterincubation, in one continuous motion, invert magnet and tube and pouroff desired fraction into a 50 mL conical. These procedures wererepeated for remaining PBMCs (per one donor) and combined. The combinedfraction was centrifuged at 1200 rpm for 5 minutes and then supernatantswere discarded and cells were resuspended in 5 mL of B cell media. Theisolated CD19+ cells were counted on the cell counter.

8.4.3 B cell Differentiation Assay

Step 1—B cell Activation—day 0 through day 4: Prepare fresh B cellcocktail by adding 50 μg/mL of human transferrin to B cell media.(Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin). Filter requiredvolume of media needed for experiment through a 0.22 μM filter. Add Bcell differentiation cocktail (final concentration): recombinant humanIL-2 (20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), CD40Ligand/TNFSF5/histidine-tagged (50 ng/mL), polyHistidine mouse IgG1antibody (5 μg/mL), and ODN 2006-Human TLR9 ligand (10 μg/mL) to cells.Five milliliters (1×10⁵/ml) of CD19+B cell were added to each well of a6 well flat-bottom plate (final cell count=5×10⁵/well). Five μL(1×)±compound/DMSO was added to each test well (0.1% final DMSO) andincubated at 37

° C. for 4 days.

Step 2—Plasmablast Generation—day 4 through day 7: Cells were harvestedand counted on the cell counter; an aliquot was removed for flowanalysis, the remaining cells were washed with PBS. Prepare fresh B cellcocktail by adding 1 μg/ml of human transferrin to B cell media.(Iscoves+10% PFBS, 1% P/S, and 5 μg/mL human insulin Filter requiredvolume of media needed for experiment through a 0.22 filter. Add B celldifferentiation cocktail (final concentration): recombinant human IL-2(20 U/mL), IL-10 (50 ng/mL), IL-15 (10 ng/mL), IL-6 (50 ng/mL) to cells.Add fresh B cell cocktail and transfer cells back to the original wellsand bring volume back to 5 mL. Five μL (1×)±compound/DMSO was added toeach test well (0.1% final DMSO) and incubated at 37

° C. for 4 days.

On day 7, cells were harvested and counted on the cell counter. Cellswere then divided for flow analysis and the remaining cells were lysedwith RLTbuffer and stored at −80° C. for RNA extraction and geneexpression. Supernatants were aliquoted and frozen at −20° C. forimmunoglobulin assays.

8.4.4 Preparation of Test Compound Stock Solutions and Dilutions

The test compounds was weighed and dissolved in sterile 100% DMSO(dimethyl sulfoxide; Research Organics, Cleveland, Ohio) to create 40 mMstock solution. Dilutions of the 40 mM stock were used in the assay toobtain final test compound concentrations based on experimental design.

8.4.5 RNA Extraction and Gene Expression

Differentiated B cells (see section 4.3.3) were harvested for totalribonucleic acid (RNA) preparation with a Qiacube RNA extractioninstrument (Qiagen, Valencia, Calif.) using QIAGEN RNeasy minispin-column kits. Purified RNA was reverse transcribed into cDNA withthermal cycler [MJ Research; Inc., St. Bruno, Quebec, Canada) using areverse-transcriptase kit (Applied Biosystems). The gene expressionassay was carried out using 7500 RT-PCR system (Applied Biosystems) intriplicate. A glyceraldehyde 3-phosphate dehydrogenase gene expressionassay control was run for each sample and used as a normalizationcontrol. For each gene, samples within each experiment were normalizedto 0.1% DMSO treatment only for that particular time point.

Supernatants (from section 8.4.3) were harvested and analyzed by ELISAfor IgG and IgM production (ZeptoMetrix Corp. Buffalo, N.Y.).

8.4.6 Cell Phenotyping

Differentiated B cells (see section 4.3.3) were harvested, counted, andaliquoted at about 1×10⁶ cells or less per 4 mL tube. The cells werewashed 1× with stain buffer. Next, the cells then were blocked with 10%human serum/PBS for 20-30 minutes. Following blocking, the cells werecentrifuged for 5 minutes at 1200 rpm and supernatants discarded. In the100 μL of remaining buffer, 20 μL of various BD Pharmigen flowantibodies were added according to experimental design. The cells werestained for 20-30 minutes at 4° C. Then the cells were washed 2× withstain buffer and supernatants discarded. Next, 500 μL of stain buffer orPBS was added to the tubes. The samples were immediately analyzed or putat 4° C. overnight. Cells were stained with mouse anti-human CD20 andCD38, CD19 and CD27, or respective isotype controls. All samples wereanalyzed using a FACSCanto flow cytometer, FACSDiva analysis software(BD Bioscience), and FlowJo Analysis software.

8.4.7 Cell Viability Analysis

To determine live cell count, B cells (see section 4.3.3) were stainedwith 0.4% trypan blue and live cells counted using the Countessautomated cell counter (Invitrogen) in duplicate samples.

The data was graphed using GraphPad Prism 5.0 software. IC₅₀ values werecalculated using non-linear regression, sigmoidal-dose responseconstraining the top to 100% and bottom to 0% allowing for a variableslope. The results for test compounds in the Ig assays were expressed asthe percentage inhibition relative to control DMSO values.

The potency for inhibition of normal and SLE PBMC production of IgG andIgM for the test compound is as follows:

IgG IC₅₀ (nM) IgM IC₅₀ (nM) SLE (n = 3) Normal (n = 3) SLE (n = 3)Normal (n = 3) 46 61 30 23

8.5 Example 5 Effects on Prevention and Treatment of Bleomycin InducedDermal Fibrosis

In this example, the effects of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione (testcompound) on the progression of experimental fibrosis and the regressionof established fibrosis in a mouse model of bleomycin-induced dermalfibrosis was studied.

The following abbreviations are used in this example:

Abbreviation or Specialist Term Explanation or Definition ANOVA Analysisof Variance α-SMA Alpha Smooth Muscle Actin CMC Carboxymethyl CelluloseECM Extracellular Matrix NaCl Sodium Chloride PO orally QD Once dailydosing SSc Systemic Sclerosis

DBA/2 mice were used in this study. Eight animals were used pertreatment group in the study.

Mice were kept in the animal house under standard conditions with foodand water ad libidum.

The vehicle, 0.5% carboxymethyl cellulose (CMC)/0.25% Tween 80, wasprepared in distilled H₂O and dissolved overnight on a magnetic stirrer(add 0.5 g CMC; Sigma #C9481) and 0.25 ml Tween 80 (Sigma #P8074) to99.75 ml to make a total of 100 ml 0.5% CMC/0.25% Tween 80).

The test compound powder was weighed out and suspended fresh daily inthe vehicle 0.5% CMC/0.25% Tween 80, to avoid drug hydrolysis in theaqueous medium. The compound was suspended, not dissolved, in thisvehicle. The formulation was homogenized with a Teflon pestle and mortar(Potter-Elvehjem tissue grinder) using a motorized Eberbach tissuehomogenizer. The daily drug stock concentration used in these studieswas 3 mg/ml.

Bleomycin was obtained from the pharmacy of the University ofErlangen-Nuremberg and freshly prepared once a week. Skin fibrosis wasinduced in 6-week-old DBA mice by local intracutaneous injections of 100μl of bleomycin dissolved in 0.9% NaCl, at a concentration of 0.5 mg/ml,every other day in defined areas of 1.5 cm2 on the upper back.

Study Design

The mouse model of bleomycin induced dermal fibrosis is widely used toevaluate anti-fibrotic therapeutics. In this model, a localized dermalfibrosis is induced by intradermal injections with bleomycin every otherday for 3 weeks. This model resembles early, inflammatory stages of SSc.To evaluate potential effects on prevention of fibrosis, treatment wasinitiated simultaneously with the first bleomycin injection. To studythe effect of test compound on prevention of bleomycin-induced dermalfibrosis in vivo, the treatments were divided into following groups:

-   -   Control group: Intradermal injection of NaCl for 3 weeks.        Treatment consisted of administration of the vehicle (0.5%        CMC/0.25% Tween 80).    -   Untreated bleomycin group: Intradermal injection of bleomycin        for three weeks. Administration of the vehicle (0.5% CMC/0.25%        Tween 80).    -   Test compound group: Intradermal injection of bleomycin for        three weeks. The test compound was administered at 30 mg/kg; PO,        QD.    -   Positive control group: Intradermal injection of bleomycin for        three weeks. Injection of Imatinib (50 mg/kg; IP, QD). Imatinib        mesylate has previously been shown to exert potent anti-fibrotic        effects in bleomycin induced dermal fibrosis. See Akhmetshina A.        et al., Arthritis Rheum 2009; 60(1):219-224.

To evaluate regression of fibrosis, a modified model of bleomycininduced dermal fibrosis was used. Mice were pre-challenged withbleomycin to induce a robust skin fibrosis. One group received treatmentwith the test compound, while challenge with bleomycin was ongoing foradditional three weeks. The outcome of this group was compared to micechallenged with bleomycin for six weeks (prevention of furtherprogression) and to mice challenged with bleomycin for three weeksfollowed by NaCl for additional three weeks (induction of regression).The following groups were used in the regression study:

-   -   Control group: Intradermal injection of NaCl for six weeks.        Control treatment consisted of administration of the vehicle.    -   Untreated bleomycin group 1 (regression): Intradermal injection        of bleomycin for three weeks followed by intradermal injections        of NaCl for another three weeks. Treatment consisted of        administration of the vehicle. Untreated bleomycin group 2        (prevention of progression): Intradermal injection of bleomycin        for six weeks. Treatment consisted of administration of the        vehicle.    -   Test compound group: Intradermal injection of bleomycin for six        weeks. The test compound was administered at 30 mg/kg; PO, QD.    -   Positive control group: Intradermal injection of bleomycin for        six weeks. Injection of Imatinib (50 mg/kg; IP, QD)

Experimental Procedure

Dermal thickness was determined by staining with hematoxylin and eosinand activated fibroblasts by using immunohistochemistry for alpha smoothmucle actin (α-SMA). The dermal thickness, as determined by the modifiedRodnan Skin Score, is currently the most common primary outcome in humanclinical trials for anti-fibrotic agents in SSc. Skin sections werestained with hematoxylin/eosin for better visualization of the tissuestructure. Dermal thickness was analyzed with a Nikon Eclipse 80imicroscope (Nikon, Badhoevedorp, The Netherlands) by measuring themaximal distance between the epidermal-dermal junction and thedermal-subcutaneous fat junction at 4 different skin sections in eachmouse. The evaluation was performed by 2 independent examiners.

For quantification of myofibroblasts, skin sections were deparaffinizedand incubated with 5% bovine serum albumin for 60 minutes. Cellspositive for α-SMA were detected by incubation with monoclonalanti-α-SMA antibodies (clone 1A4; Sigma-Aldrich, Steinheim, Germany) for2 hours at room temperature followed by incubation with 3% hydrogenperoxide for 10 minutes. Goat anti-rabbit antibodies labeled withhorseradish peroxidase (Dako, Hamburg, Germany) were used as secondaryantibodies. The expression of α-SMA was visualized with3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Monoclonalmouse IgG antibodies (Calbiochem, San Diego, Calif.) were used ascontrols.

In addition, the amount of collagen in lesional skin will be measuredwith the SirCol collagen assay; RNA and plasma of all mice were savedfor further analyses.

The test compound significantly decreases dermal thickness of lesionalskin in the bleomycin dermal fibrosis mouse model. The test compound at30 mg/kg; PO, QD significantly prevented dermal thickening byapproximately 22±0.49% (p<0.0001).

Representative photomicrographs of hematoxylin and eosin stained skinsections are shown in FIG. 8. Dermal thickness was assessed by measuringthe maximal distance between the epidermal-dermal junction and thedermal-subcutaneous fat junction. The line drawn between the junctionpoints shows the relative thickness in the treatment groups.

To determine the effect of the treatments on fibroblast activation,α-SMA+myofibroblasts were counted in lesional skin sections. The testcompound at 30 mg/kg; PO, QD reduced the number of myofibroblasts by30±0.23% (p<0.0001).

Effect on the Regression of Bleomycin Induced Dermal Fibrosis

The inhibitory effects of the test compound on progression of fibrosiswere also confirmed in the modified bleomycin model designed toinvestigate potential regression of fibrosis. The test compound reduceddermal thickness of bleomycin induced dermal thickening by 22±0.28%(p<0.0001). FIG. 9 shows photomicrographs of representative hematoxylinand eosin stained skin sections. Dermal thickness was assessed bymeasuring the maximal distance between the epidermal-dermal junction andthe dermal-subcutaneous fat junction. The line drawn between thejunction points shows relative thickness in the treatment groups.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1. A method for treating, preventing or managing an immune-relateddisease or an inflammatory disease comprising administering to a patientin need thereof an effective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer, racemic mixture, co-crystal, clathrate, or polymorph thereof.2. The method of claim 1, wherein the disease is systemic lupuserythematosus, scleroderma, Sjögren syndrome, ANCA-induced vasculitis,anti-phospholipid syndrome or myasthenia gravis.
 3. The method of claim1, wherein the disease is systemic lupus erythematosus.
 4. The method ofclaim 1, wherein the disease is severe systemic lupus erythematosus. 5.The method of claim 1, wherein the disease is scleroderma.
 6. The methodof claim 5, wherein the scleroderma is localized, systemic, limited ordiffuse scleroderma.
 7. The method of claim 6, wherein the systemicscleroderma comprises CREST syndrome.
 8. A method for reducing,inhibiting or preventing a symptom of systemic lupus erythematosuscomprising administering an effective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer or racemic mixture thereof to a patient having the symptom ofsystemic lupus erythematosus, wherein the symptom is selected from thegroup consisting of joint pain, joint swelling, arthritis, chest painwhen taking a deep breath, fatigue, fever with no other cause, generaldiscomfort, uneasiness, hair loss, mouth sores, swollen lymph nodes,sensitivity to sunlight, skin rash, headaches, numbness, tingling,seizures, vision problems, personality changes, abdominal pain, nausea,vomiting, abnormal heart rhythms, coughing up blood and difficultybreathing, patchy skin color and Raynaud's phenomenon.
 9. A method forreducing, inhibiting or preventing a symptom of scleroderma comprisingadministering to a patient having the symptom of scleroderma aneffective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer, racemic mixture, co-crystal, clathrate, or polymorph thereof,wherein the symptom is selected from the group consisting of (i) gradualhardening, thickening, and tightening of the skin; (ii) skindiscoloration; (iii) numbness of extremities; (iv) shiny skin; (v) smallwhite lumps under the surface of the skin that erupt into a chalky whitefluid; (vi) Raynaud's esophagaeal dysfunction; (vii) telangiectasia;(viii) pain and/or stiffness of the joints; (ix) swelling of the handsand feet; (x) itching of the skin; (xi) stiffening and curling of thefingers; (xii) ulcers on the outside of certain joints, such as knucklesand elbows; (xiii) digestive problems, such as heartburn, difficulty inswallowing, diarrhea, irritable bowel, and constipation; (xiv) fatigueand weakness; (xv) shortness of breath; (xvi) arthritis; (xvii) hairloss; (xviii) internal organ problems; (xix) digital ulcers; and (xx)digital auto-amputation.
 10. A method for improving the modified Rodnanskin score, reducing or improving the skin thickness, reducing orimproving skin induration, improving the pulmonary function, improvingthe dermatology quality of life index, improving the carbon monoxidediffusing capacity, improving the Mahler Dyspnea index, improving theSaint George's Respiratory Questionnaire score, improving the UCLAscleroderma clinical trial consortium gastrointestinal tract score,improving flow-mediated dilatation or improving or increasing the sixminute walk distance of a patient having scleroderma, comprisingadministering to the patient an effective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,tautomer, racemic mixture, co-crystal, clathrate, or polymorph thereof.11. The method of claim 1 further comprising administering a secondactive agent, wherein the second active agent is an anti-inflammatory orimmunomodulatory compound.
 12. The method of claim 1, wherein theeffective amount is about 0.005 mg/kg to about 10 mg/kg of the patient'sbody weight.
 13. A method for modulating activity of a B cell comprisingcontacting the cell with an effective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione or apharmaceutically acceptable salt, solid form, solvate, hydrate,stereoisomer, tautomer, racemic mixture, co-crystal, clathrate, orpolymorph thereof thereof.
 14. A method for modulating activity of a Tcell comprising contacting the cell with an effective amount of3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione or apharmaceutically acceptable salt, solid form, solvate, hydrate,tautomer, stereoisomer, racemic mixture, co-crystal, clathrate, orpolymorph thereof thereof.
 15. The method of claim 8 further comprisingadministering a second active agent, wherein the second active agent isan anti-inflammatory or immunomodulatory compound.
 16. The method ofclaim 8, wherein the effective amount is about 0.005 mg/kg to about 10mg/kg of the patient's body weight.
 17. The method of claim 9 furthercomprising administering a second active agent, wherein the secondactive agent is an anti-inflammatory or immunomodulatory compound. 18.The method of claim 9, wherein the effective amount is about 0.005 mg/kgto about 10 mg/kg of the patient's body weight.
 19. The method of claim10 further comprising administering a second active agent, wherein thesecond active agent is an anti-inflammatory or immunomodulatorycompound.
 20. The method of claim 10, wherein the effective amount isabout 0.005 mg/kg to about 10 mg/kg of the patient's body weight.